Identifying stabilizers of p53 using pocket complementarity. The tumor suppressor protein p53 is mutated or deleted in more than half of human cancers. The most frequently occurring of these loss-of-function mutations are localized to the p53 core domain, but do not involve surface residues directly responsible for function. Rather, these point mutants reduce the thermodynamic stability of this marginally stable protein, such that cellular activity is diminished because an insufficient amount of p53 is correctly folded The goal of this proposal is to identify compounds that potently bind and stabilize correctly folded p53. We expect that stabilization through this mechanism will restore activity to this most frequently occurring class of p53 point mutants, and further will restore activity to these destabilized mutants - regardless of precisely which mutation is responsible for the underlying loss of protein stability. Already we have identified several stabilizing compounds from a small pilot screen, and we find that these compounds can restore transcriptional activity in cell lines harboring destabilized mutants of p53. Our central hypothesis is that by extending the scope of our computational studies and optimizing the resulting hit compounds through medicinal chemistry, we will identify compounds that act even more potently. We propose to meet this objective through pursuit of the following specific aims: 1) Use cutting-edge computational methods to identify compounds that bind to p53. 2) Test predicted hits in vitro using direct stability assays. 3) Optimize validated hits using structure-guided medicinal chemistry. Conventional approaches to identify compounds that stabilize p53 by binding to new surface sites might entail structure-based virtual screening, coupled with biochemical screening of the predicted hits. Each of these approaches would be expected to encounter particular hurdles when applied to this problem: the primary innovations in the proposed research lie in our use of newly-developed tools from the Karanicolas and Fisher labs to address each of these specific challenges. Using these tools we expect to identify a set of novel p53 re-activators, which in turn may represent a starting point for developing a new class of broad-spectrum cancer therapeutics. We further expect that refinement of our screening platform through these studies of p53 will additionally enhance its utility for identifying re-activators of other select proteinsthat are frequently deactivated in human cancers by destabilizing mutations.